Genetic engineering of plants has become possible by virtue of two discoveries: first of all the possibility of transformation of heterologous genetic material to the plant cell (most efficiently done by the bacterium Agrobacterium tumefaciens or related strains) and secondly by the existence of plant promoters which are able to drive the expression of said heterologous genetic material.
A typical plant promoter consists of specific elements. A basis is formed by the minimal promoter element, which enables transcription initiation, often accompanied by a sequence, also denominated as the TATA-box, which serves as a binding place for transcription initiation factors. In most promoters; the presence of this TATA-box is important for proper transcription initiation. It is typically located 35 to 25 basepairs (bp) upstream of the transcription initiation site. Another part of the promoter consists of elements which are able to interact with DNA-binding proteins. Known are G-box binding elements which are based on the hexanucleotide CACGTG motif. These elements have been shown to be able to interact with bZIP DNA-binding—proteins which bind as dimers (Johnson & McKnight, Ann. Rev. Biochem, 58, 799–839, 1989). Other G-box related motifs, such as the Iwt and PA motifs have been described (WO 94/12015).
These motifs have been shown to be involved in tissue-specific promoter expression in plants. For instance, presence of Iwt tetramers confer embryo-specific expression, while PA tetramers confer high level root expression, low-level leaf expression and no seed expression.
Similarly, GT-1 like binding sites (grouped on basis of a moderate consensus sequence GGTA/TA) are described. Such a binding site is found far upstream the promoter region of the Arabidopsis plastocyanin promoter and seems to be involved in activation of transcription during light periods (Fisscher, U. et al., Plant Mol. Biol. 26, 873–886, 1994).
Another sequence-related phenomenon which is found often in plant promoters is the presence of sequences which enable the formation of Z-DNA. Z-DNA is DNA folded in a left-handed helix which is caused by repeats of dinucleotides GC or AC. It is believed that folding in a Z-form influences the availability of the DNA for approach by RNA polymerase molecules, thus inhibiting the transcription rate.
One of the earliest and most important inventions in the field of plant protein expression is the use of (plant) viral and Agrobacterium-derived promoters that provide a powerful and constitutive expression of heterologous genes in transgenic plants. Several of these promoters have been used very intensively in plant genetic research and still are the promoter of choice for rapid, simple and low-risk expression studies. The most famous are the 35S and 19S promoter from Cauliflower Mosaic Virus (CaMV), which was already found to be practically useful in 1984 (EP 0 131 623), the promoters which can be found in the Agrobacterium T-DNA, like the nopaline synthase (nos), mannopine synthase (mas) and octopine synthase (ocs) promoters (EP 0 122 791, EP 0 126 546, EP 0 145 338). A plant-derived promoter with similar characteristics is the ubiquitin promoter (EP 0 342 926).
In time, several attempts have been made to increase the level of expression of these promoters. Examples for this are the double enhanced 35S promoter (U.S. Pat. No. 5,164,316) and, more recently, the superpromoter, which couples parts of the Agrobacterium promoters (EP 729 514).
However, in many cases these promoters do not fulfill the criteria of an ideal promoter. All promoters described above show a clear pattern of organ- or developmental-specific expression, and frequently the pattern of expression found with these promoters is not ideal for some applications. Especially for biotechnological applications like the engineering of fungal and insect resistance, which require expression both in the right location as well as in the right timeframe of plant development there is a need for new constitutive promoters which are able to give a high level of transgene expression at exactly the right time and place.